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Specific inhibition of expression of a human collagen gene (COL1A1) with modified antisense oligonucleotides. The most effective target sites are clustered in double-stranded regions of the predicted secondary structure for the mRNA.
Laptev, A. V.; Lu, Z.; Colige, Alain et al.
1994In Biochemistry, 33 (36), p. 11033-9
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Keywords :
3T3 Cells; Animals; Base Sequence; Binding Sites; Collagen/genetics/metabolism; DNA Primers; Gene Expression Regulation/drug effects; Humans; Mice; Molecular Sequence Data; Nucleic Acid Conformation; Oligonucleotides, Antisense/pharmacology; RNA, Messenger/chemistry/drug effects
Abstract :
[en] A series of antisense oligonucleotides (ASOs) were synthesized and tested to define the best target sites within an RNA transcript of collagen for effective inhibition of expression. The test system consisted of mouse NIH 3T3 fibroblasts that were stably transfected with a human minigene for procollagen I so that the cells simultaneously synthesized full-length mouse pro alpha 1 (I) chains and internally deleted human pro alpha 1 (I) chains. The sequences of the transcripts from both genes were compared, and a series of 28 ASOs were designed to target sites in which there were at least two base differences within a 20-nucleotide sequence between the human and mouse transcripts. Six of the ASOs specifically decreased the levels of pro alpha 1 (I) chain synthesized from the human gene without a decrease in the levels of pro alpha 1 (I) chains from the mouse endogenous gene. The most effective ASOs reduced the intracellular levels of human pro alpha 1 (I) chains relative to the mouse pro alpha 1 (I) chains to 37-67% of the control values. Combined addition of two effective ASOs or a second administration of the same effective ASO did not produce any additive effect. The results did not support previous suggestions that the best target sites for ASOs were sequences around initiation codons for translation, at intron-exon boundaries, or in single-stranded loops in hairpin structures. Also, the results did not support previous suggestions that the most effective ASOs are those with the highest affinities for their target sequences. Instead, the most consistent pattern in the data was that the most effective ASOs were those targeted to sequences that were predicted to form clustered double-stranded structures in RNA transcripts.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Laptev, A. V.;  Thomas Jefferson University, Philadelphia, Pennsylvania > Department of Biochemistry and Molecular Biology > Jefferson Institute of Molecular Medicine, Jefferson Medical College
Lu, Z.;  Thomas Jefferson University, Philadelphia, Pennsylvania > Department of Biochemistry and Molecular Biology > Jefferson Institute of Molecular Medicine, Jefferson Medical College
Colige, Alain ;  Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Laboratoire des tissus conjonctifs
Prockop, D. J.;  Thomas Jefferson University, Philadelphia, Pennsylvania > Department of Biochemistry and Molecular Biology > Jefferson Institute of Molecular Medicine, Jefferson Medical College
Language :
English
Title :
Specific inhibition of expression of a human collagen gene (COL1A1) with modified antisense oligonucleotides. The most effective target sites are clustered in double-stranded regions of the predicted secondary structure for the mRNA.
Publication date :
1994
Journal title :
Biochemistry
ISSN :
0006-2960
eISSN :
1520-4995
Publisher :
American Chemical Society, Washington, United States - District of Columbia
Volume :
33
Issue :
36
Pages :
11033-9
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 18 June 2010

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